Voltage-activated sodium channels are responsible for the fast depolarizing phase of the action potential that underlies electrical signaling in neurons, muscles and other electrically excitable cells (reviewed by Hille, 1992 Ionic Channels of Excitable Membranes (Sinauer, Sunderland, Mass.)). Biochemical characterization of voltage-activated sodium channels from a variety of tissues indicate that they all contain a single alpha subunit of molecular weight ranging from 230,000 to 300,000 (reviewed by Catterall, 1992 Cellular and Molecular Biology of Voltage-gated Sodium Channels. Physiological Reviews, 72:S15-S48). The alpha subunit of the Electrophorus electricus voltage-activated sodium channel was cloned using biochemical and molecular genetic techniques (Noda, et al., 1984 Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature, 312:121-127.). The purified Electrophorus electricus sodium channel alpha subunit forms a functional voltage-activated sodium channel as a single alpha subunit (Rosenberg, R. L., et al., 1984, Proc. Natn. Acad. Sci. U.S.A. 1:1239-1243). The cDNA encoding the Electrophorus electricus voltage-activated sodium channel was used to isolate cDNAs encoding three distinct, but highly homologous rat brain voltage-activated sodium channel genes (Kayano et al., 1988, Primary structure of rat brain sodium channel III deduced from the cDNA sequence, FEBS Lett. 228:187-194; Noda et al. 1986, Nature .320;188-192). Biochemical analysis of voltage-activated sodium channels from rat brain indicate that the alpha subunits am associated noncovalently with a beta1 subunit (36,000 kDa) and are disulfide linked to a beta2 subunit (33,000 kDa) which is not required for channel activity (Hartshorne and Catterall, 1981, Purification of the saxitoxin receptor of the sodium channel from rat brain. Proc. Natl. Acad. Sci. U.S.A. 78:4620-4624; Hartshorne and Catterall 1984, The sodium channel from rat brain. Purification and subunit composition. J. Biol. Chem. 259:1667-1675; Hartshorne, et al., 1982, The saxitoxin receptor of the sodium channel from rat brain. Evidence for two nonidentical beta subunits. J. Biol. Chem. 257:13888-13891; Messsner and Catterall, 1985, The sodium channel from rat brain. Separation and characterization of subunits. J. Biol. Chem. 260:10597-10604). RNAs transcribed from cDNAs encoding alpha subunits of mammaliam voltage-activated sodium channels are sufficient to direct the synthesis of functional sodium channels when injected into Xenopus oocytes (Auld et al. 1988, A rat brain Na+channel alpha subunit with novel gating properties. Neuron 1:448-461; Moorman et al. 1990, Changes in sodium channel gating produced by point mutations in a cytoplasmic linker. Science 250:688-691; Noda et al. 1986, Expression of functional sodium channels from cloned cDNA. Nature 322:826-828; Suzuki et al. 1988, Functional expression of cloned cDNA encoding sodium channel III. FEBS Lett. 228:195-200). Although alpha subunits of mammalian voltage-activated sodium channels are sufficient to encode functional sodium channels in Xenopus oocytes, their biophysical properties are not identical to those observed in intact cells. Co-expression of the rat brain voltage-activated sodium channel beta1 subunit with the rat brain type IIa alpha subunit in Xenopus oocytes restores the normal biophysical properties observed in intact cells (Isom et al. 1992, Primary structure and functional expression of the B 1 subunit of the rat brain sodium channel. Science 256: 839-842).
Biochemical characterization of insect neuronal sodium channels has revealed that they contain an alpha subunit of molecular weight ranging from 240,000 to 280,000, but they lack any covalently linked beta subunits (Gordon et al 1993, Biochemical Characterization of Insect Neuronal Sodium Channels. Archives of Insect Biochemistry and Physiology 22:41-53). Partial DNA sequences from the fruit fly Drosophila melanogaster presumed to encode voltage-activated sodium channels were initially identified on the basis of homology to vertebrate voltage-activated sodium channel alpha subunits (Salkoff et al. 1987, Genomic organization and deduced amino acid sequence of a putative sodium channel genes in Drosophila. Science 237:744-749; Okamoto et al. 1987, Isolation of Drosophila genomic clones homologous to the eel sodium channel gene. Proc. Jpn. Acad. 63(B):284-288; Ramaswami and Tanouye, 1989, Two sodium-channel gene in Drosophila: Implications for channel diversity. Proc. Natn. Acad. Sci. U.S.A. 86:2079-2082). Using a molecular genetic approach it was determined that the paralytic (para) locus in Drosophila encodes a voltage-activated sodium channel alpha subunit and the entire para cDNA sequence was determined (Loughney et al. 1989, Molecular analysis of the para locus, a sodium channel gene in Drosophila. Cell 58:1143-1154;Thackeray and Ganetzky 1994, Developmentally regulated alternative splicing generates a complex array of Drosophila para sodium channel isoforms. J. Neuroscience 14:2569-2578).
It has been proposed that the Drosophila ripe locus encodes a regulatory or structural component of voltage-activated sodium channels for the following reasons: (1) [3H] saxitoxin binding to voltage-activated sodium channels is reduce 30-40% in tipE mutants (Jackson et al. 1986, The tipE mutation of Drosophila decreases saxitoxin binding and interacts with other mutations affecting nerve membrane excitability. J. of Neurogenetics, 3:1-17), (2) sodium current density is reduced 40-50% in cultured embryonic neurons from tipE mutants (O'Dowd and Aldrich, 1988, Voltage-Clamp Analysis of Sodium Channels in wild-type and Mutant Drosophila Neurons. J. of Neuroscience, 8:3633-3643), (3) para;tipE mutants exhibit unconditional lethality in an allele specific manner (Ganetzky 1986, Neurogenetic analysis of Drosophila Mutations affecting Sodium Channels: Synergistic Effects on Viability and Nerve Conduction in Double Mutants involving tipE. J. of Neurogenetics, 3:19-31. Jackson et al. 1986, The tipE mutation of Drosophila decreases saxitoxin binding and interacts with other mutations affecting nerve membrane excitability. J. of Neurogenetics, 3:1-17), (4) para and tipE RNA are expressed in the embryonic CNS and PNS (Hall et al. 1994, Molecular and genetic analysis of tipE: a mutation affecting sodium channels in Drosophila. Presented at the 351h Annual Drosophila Research Conference, Apr. 20-24, 1994, Chicago, Ill.; Hong and Ganetzky 1994, Spatial and temporal expression patterns of two sodium channel genes in Drosophila. J. Neuroscience, 14:5160-5169), (5) ripe encodes a 50kDa acidic protein with two putative membrane spanning domains, a membrane topology shared by other ion channel subunits (Hall et al. 1994, Molecular and genetic analysis of tipE: a mutation affecting sodium channels in Drosophila. Presented at the 351h Annual Drosophila Research Conference, Apr. 20-24, 1994, Chicago, Ill.; Hall and Feng 1994, The ripe locus defines a novel membrane protein required during development to rescue adult paralysis. Presented at the 481h annual meeting of the Society of General Physiologists, Sep. 7-11, 1994, Woods Hole Mass.). The Drosophila ripe locus has been cloned and sequenced but the nucleotide and amino acid sequence of ripe are presently undisclosed (Hall et al. 1994, Molecular and genetic analysis of tipE: a mutation affecting sodium channels in Drosophila. Presented at the 351h Annual Drosophila Research Conference, Apr. 20-24, 1994, Chicago, Ill.; Hall and Feng 1994, The tipE locus defines a novel membrane protein required during development to rescue adult paralysis (para). Presented at the 481h annual meeting of the Society of General Physiologists, Sep. 7-11, 1994, Woods Hole Mass.).